Osteogenic Composite Matrix, Method for the Production Thereof and Implant and Scaffold for Tissue Engineering Provided with a Coating Formed by Said Osteogenic Composite matrix

ABSTRACT

The invention relates to an osteogenic composite matrix consisting of collagen and non-collagen components of an extracellular matrix (ECM-components), to a method for producing said matrix, to a method for producing an implant or a scaffold for tissue engineering which is provided with a coating formed by said osteogenic composite matrix and is used for stimulating and accelerating a hard tissue formation such as, for example the implant osseointegration in bones. The inventive osteogenic composite matrix comprises a collagen and at least one non-collagen ECM component or the derivatives thereof, wherein the collagen component consists of non-crosslinked collagen fibres produced by fibrillogenesis and the non-collagen ECM component or the derivatives thereof are integrated into said collagen fibres.

TECHNICAL FIELD

The invention relates to an osteogenic matrix composite of collagen andnoncollagenic components of the extracellular matrix (ECM components), amethod for its production, a method for the production of an implant orof a scaffold for tissue engineering having a coating of an osteogenicmatrix composite, and implants and scaffolds for tissue engineeringhaving a coating of the osteogenic matrix composite for the stimulationand accelerated formation of hard tissue, such as, for example, in thefield of osseointegration of implants into bone.

BACKGROUND ART

In the tissue, the cells are embedded in the native extracellular matrix(ECM), which is an important part of the cellular environment. Thenative ECM is a highly ordered, tissue-specific network which consistsof collagens, glycoproteins, proteoglycans and glycosaminoglycans (GAG).The composition for various tissue and for various stages of developmentis very different here, such that the respective matrix has specificproperties with respect to interaction with cells and growth factors.

The main structural protein of the native bone matrix is collagen typeI, but various other matrix proteins such as proteoglycans andglycoproteins can interact with the collagen and influence the structureand function of the matrix. These noncollagenic ECM proteins fulfillspecific functions in the matrix. Thus fibronectin, in addition tocell-binding properties, also has collagen- and GAG-binding properties[Stamatoglou and Keller, 1984, Biochim Biophys Acta. Oct. 28; 719(1):90-7], whereas small leucine-rich proteins (SLRPS) such as decorin notonly play a role in the organization of the native ECM (decorinmodulates fibrilogenesis in vivo), but also bind growth factors such asTGF-β or even play a role as signal molecules [Kresse and Schönherr,2001, J Cell Phys 189: 266-274].

Proteoglycans and glycoproteins differ by their degree of glycosylation,the sugar content of the particularly highly glycosylated proteoglycansconsisting of various glycosaminoglycans. The distribution of thesechains can be tissue-specific, as, for example, for decorin (chondroitinsulfate in the bone, dermatan sulfate in the skin). Theglycosaminoglycans are large, unbranched polysaccharides which consistof repeating disaccharides, which are composed, for example, ofN-acetyl-galactosamine, N-acetylglucosamine, glucuronate or iduronate,which are sulfated to different degrees. The sugar chains are present invivo bound to the proteoglycans and play an important role in thefunction of these proteins, i.e. in growth factor binding and modulation[Bernfield et al, 1999, Annu Rev Biochem, 68: 729-771].

Individual ECM constituents, in particular collagen, are alreadyutilized for the biocompatible modification of scaffolds and implants inorder to improve cell adhesion and tissue integration. In addition tocollagen, further ECM components such as polysaccharides are used invarious applications. Thus bone tissue was crosslinked withglycosaminoglycans in order to produce a three-dimensional scaffold forapplications in tissue culture (WO 01/02030A2).

A chondroitin sulfate-containing mixture is used for the repair of bonedefects; this promotes the healing of the connective tissue, mainly onaccount of the content of aminosugars and increased matrix productioncaused thereby (WO 98/27988, WO 99/39757). In combination with collagen,plant polysaccharides are used as wound coverings (EP 0140569 A2), and acombination of chitosan and GAGs is described as an agent for thestimulation of the regeneration of hard tissue (WO 96/02259).Collagen-GAG mixtures are produced here by acid coprecipitation, anunstructured precipitate and no defined collagen fibrils comparable tothose in the native ECM being formed (U.S. Pat. No. 4,448,718, U.S. Pat.No. 5,716,411, U.S. Pat. No. 6,340,369).

With progressive availability of recombinant growth factors, thoseosteoinductive factors which actively influence the interactions betweenimplants and surrounding tissue are increasingly of interest for implantapplications [Anselme K (2000). Biomaterials. 21, 667-68]. In connectionwith bone healing, the ‘bone morphogenetic proteins’ (BMP 2, 4-7) areparticularly interesting since they induce the differentiation ofmesenchymal stem cells in chondrocytes and osteoblasts and the formationof new bone [Celeste A J, Taylor R, Yamaji N, Wang J, Ross J, Wozney J M(1994) J. Cell Biochem. 16F, 100; Wozney J M, Rosen V (1993) Bonemorphogenetic proteins in Mundy, G R, Martin T J (Ed.) Physiology andpharmacology of bone. Handbook of experimental pharmacology, Vol. 107.Springer Verlag, Berlin, 725-748]. On account of these strongbone-inducing effects, recombinant BMPs are employed in various carriermaterials in order to promote and to improve the regeneration of bone.Effective carriers for morphogenetic proteins should bind these, protectagainst hydrolysis, make possible subsequent, controlled release andpromote the associated cell reactions. Moreover, such carriers should bebiocompatible and biodegradable. Preferred carrier materials for BMPsare, for example, xenogenic bone matrix (WO 99/39757) or natural tissuesubsequently crosslinked with GAGs (WO 01/02030 A2), or HAP, collagen,TCP, methylcellulose, PLA, PGA, and various copolymers (EP 0309241 A2,DE 19890329, EP 0309241 A2, DE 19890906, WO 8904646 A1, DE 19890601).Further applications comprise a crosslinked synthetic polymer which cancontain additional components such as GAGs, collagen or bioactivefactors (WO 97/22371), or crosslinked collagen mixed withglycosaminoglycans and osteogenic factors (WO 91/18558, WO 97/21447).The collagen-GAG mixture is in this case likewise produced by acidcoprecipitation.

The use of recombinant growth factors is associated with greatdisadvantages. Since the recombinant factors usually have a loweractivity than the endogenous factors occurring naturally in the tissue,in order to achieve an effect in vivo unphysiologically high doses arenecessary. The administration of recombinant factors can only simulatethe action of endogenous factors very incompletely.

By the use of factors which promote the action of the BMPs (Bonemorphogenetic protein), or by the use of cells which can express thegrowth factors in situ, it is attempted to minimize or to circumventthis problem (WO 97/21447, WO 98/25460). Further problems can resultfrom the fact that receptors for BMP occur in many different tissues;the function of these growth factors is thus not limited to the bone.

SUMMARY OF THE INVENTION

It is the object of the present invention to specify a biocompatible andbiodegradable matrix composite which promotes and accelerates boneaccumulation and bone growth in the immediate environment and on thesurface of implants coated with the matrix composite, and which can beused in particular for the coating of synthetic, metallic or ceramicimplants. A further aim of the invention is a coating of carriermaterials (scaffolds) for tissue engineering, which assists theproduction of hard tissue in vitro and subsequently in vivo.

The invention is based on the scientific observation that for implantsin contact with the bone in most cases an adequate amount of endogenousbone-forming factors is present on account of the surrounding tissue andthe blood circulation. The bone-inducing effect of the BMPs, which canbe observed under physiological conditions in vivo, is in allprobability also not due to an individual growth factor type, but theresult of the synergistic action of a large number of endogenousfactors.

Against this background, an implant coating is desirable whichadvantageously utilizes the endogenous bone-forming factors which arepresent at the implantation site.

According to the invention, the object is achieved by an osteogenicmatrix composite of collagen and at least one noncollagenic ECMcomponent or its derivatives, in which the collagen component consistsof non-crosslinked collagen fibrils produced by means offibrillogenesis, into which are integrated the at least onenoncollagenic ECM component or its derivatives.

For the osteogenic matrix composite, according to the inventionconstituents of the extracellular matrix are used which are as similaras possible in composition and morphology to the matrix constituentswhich occur naturally in the bone, which are biocompatible andbiodegradable, and have bone tissue-specific functions both in thebinding and presentation of growth factors, and can directly influencethe reactions of the cells. As a result, a microenvironment which is asapproximate as possible to the in vivo conditions is presented to thecells, which positively influences the cell functions and the reactionto bone-forming factors such as growth factors.

The term collagen comprises all fibril-forming collagen types. Anycollagen source is suitable which produces noncrosslinked, acid-solublecollagen monomers, recombinant or tissue derived, with and withouttelopeptides.

The term noncollagenic ECM components comprises both glycosaminoglycansand noncollagenic proteins, which are known constituents of the nativeECM.

The term noncollagenic proteins comprises all matrix proteins havingnoncollagenic (proteoglycans and glycoproteins) or partly collagenic(FACITs) structure.

The main constituent of the osteogenic matrix composite is collagen oftype I, II, III, V, IX, XI, or combinations thereof. In principle, everyfibril-forming collagen type can be used which produces noncrosslinked,acid-soluble collagen monomers, collagen I, III and V being preferred,since these are the collagens mainly represented in the bone.

As GAG components, the osteogenic matrix composition containschondroitin sulfate A, C, D, E; dermatan sulfate, keratan sulfate,heparan sulfate, heparin, hyaluronic acid or their derivatives, bothindividually and mixed, chondroitin sulfate being preferred. The sugarsused are either prepared synthetically or isolated from biologicalsources.

As further noncollagenic matrix proteins, the osteogenic matrixcomposition can contain fibronectin, decorin, biglycan, laminin orversican, both individually and mixed, decorin and biglycan beingpreferred. The proteins used are either prepared recombinantly orisolated from biological sources in native form.

In order to generate a matrix which is as bone-analogous as possible,preferably collagen type I, decorin and biglycan and/or their GAG chainssuch as chondroitin sulfate are employed. Decorin or biglycan are usedhere in order to utilize bonds or synergisms between matrix, growthfactor and cell. A further possibility, which is given preference here,is the use of GAG chains, which bind endogenous growth factors or canpotentiate in their action; in particular the chondroitin sulfatefrequently occurring in the bone. By combination of collagen withfurther GAGs or matrix constituents, further endogenous growth factorscan also be used for accelerated healing, such as, for example, VEGF byheparan sulfate for the promotion of invascularization.

According to the invention, an osteogenic matrix composite of collagenand at least one noncollagenic ECM component or its derivatives isprepared such that collagen fibrils are produced by means offibrillogenesis and that prior to fibrillogenesis at least onenoncollagenic ECM component or its derivatives is added.

The collagen fibrils produced in this way can be utilized as a coatingsolution after resuspension in water or in a buffer system orlyophilized.

The fibrillogenesis (i.e. the formation of collagen fibrils) proceedsunder the following conditions: temperature range from 4° C. to 40° C.,preferably 25° C. to 37° C., collagen concentration of 50 to 5000 μg/ml,preferably 250 to 1000 μg/ml, pH 4 to pH 9, preferably pH 6 to pH 8,phosphate content up to 500 mmol/l, preferably 30 to 60 mmol/l, NaClcontent up to 1000 mmol/l, preferably up to 300 mmol/l.

By means of the preparation method according to the invention, anosteogenic matrix composite is formed having a defined structure andcomposition comparable to the situation in the native ECM.

An ordered, mutually transposed lateral association of the collagenmonomers is characteristic of collagen fibrils in vivo, a typical bandpattern having a periodicity of 64 to 67 nm resulting. This associationis due, inter alia, to the charge pattern of the monomers. Fibrilformation in vitro is induced by the pH, the temperature and the ionicstrength of a cold, acidic collagen solution being brought to values inthe vicinity of the physiological parameters.

Glycosaminoglycans or other matrix components are added to the solutioncontaining collagen monomers before fibrillogenesis and thereby includedin the following process of fibrillogenesis. Owing to the presence ofthe noncollagenic ECM components during the fibrillogenesis, these areintegrated into the resulting fibril and a matrix is formed whichcorresponds to the native ECM with respect to the components used, thecomposition and structure.

During fibrillogenesis in vitro, collagen forms the characteristictransversely striated fibrils analogously to the in vivo-structures, thestructure of the resulting fibrils being influenced by the processparameters (pH, ionic strength, phosphate concentration) and by thenature and amount of the noncollagenic components present in thereaction solution. For in vivo matrix-modifying proteoglycans such asdecorin, the greatest possible approximation to the native biologicalfunction is obtained in this way, as they can in this way also influencethe structure of the resulting fibrils under in vitro conditions.

In contrast to structure formation, as a result of aggregation byfibrillogenesis collagen aggregation can also be induced by the additionof a polyanion, as the glycosaminoglycans represent, in the acidicmedium, the electrostatic interactions existing between the GAG and thecollagen monomer being causal. In such an acid precipitate, theassociation of the collagen monomers cannot be compared with that underapproximately physiological conditions. Either an amorphous precipitateis formed or, with appropriate quantitative ratios and sufficientagreement of the charge patterns, a polymorphous aggregate such assegment long-spacing crystallites is formed.

For glycoproteins or proteoglycans such as decorin, there is nopossibility of precipitation from the acidic medium.

In order to remain as close as possible to the conditions in vivo,according to the invention the collagen fibrils are not crosslinked.Although crosslinking would increase the stability, it woulddisadvantageously have an effect on those domains which can enter intospecific bonds with endogenous bone-forming factors. This is inparticular of importance for the function of the GAGs, since theirgrowth factor-binding properties are based on free mobility of the sugarchain, which is restricted by the crosslinking. At the same time, thesugars can thus be released from the matrix, which is of importance forthe presentation of the growth factors to the cell surface.

The invention comprises the use of the osteogenic matrix compositeaccording to the invention for the coating of implants or scaffolds fortissue engineering.

Implants in the sense of the invention is understood as meaning allmetallic, ceramic and polymeric implants or implants composed of variousgroups of materials whose surfaces are at least partly in contact withbone tissue. Likewise all metallic, ceramic and polymeric structures orstructures composed of various groups of materials which serve as ascaffold for the tissue engineering of hard tissue.

The previously described osteogenic matrix composite is suitable, inparticular, for the coating of nondegradable implants in bone contact,such as artificial hip joints, tooth implants or other load-bearingapplications for which a rapid and solid integration of the implant intothe bone is necessary.

The osteogenic matrix in -combination with a three-dimensional,degradable implant, which is implanted as a bone replacement, canadvantageously accelerate the integration and the reconstruction of theimplant and also the new bone formation. These implants can contain, forexample, particulate or three-dimensional structures consisting ofcalcium phosphates, but also polymeric materials, as a basic component.

For tissue engineering, the osteogenic matrix composition in combinationwith a scaffold can be advantageous for proliferation anddifferentiation of the bone-forming cells. As a scaffold, allthree-dimensional, porous structures of synthetic and/or naturalpolymers (e.g. collagen), ceramic or metal individually or incombination are possible, biodegradable scaffolds of polymer and/orceramic being given preference.

By means of the osteogenic matrix composite, bone-forming factors, suchas, for example, growth factors which are present in vivo, are bound tothe surface of the implant after implantation and their activity isincreased. Advantageously, different endogenous factors which arepresent at the implantation site are recruited by the implant coatedwith the osteogenic matrix composite.

For the production of an implant or of a scaffold for tissueengineering, the coating solution comprising the osteogenic matrixcomposite is utilized in order to immobilize the osteogenic matrixcomposite on its surface advantageously by means of a dip-coatingprocess. The collagen concentration of the coating solution can bebetween 0.5 mg/ml to 5 mg/ml, 1 mg/ml to 2 mg/ml being the preferredrange. The osteogenic matrix composite is immobilized by incubation ofthe implant at room temperature for 5 to 20 minutes, subsequently driedand washed with water. The thickness of the resulting layer can beinfluenced by the concentration of the coating solution and by thenumber of process repetitions.

For the generation of a coated three-dimensional scaffold in combinationwith the described osteogenic matrix composite, the component mixture isadvantageously introduced into the scaffold, which can be of metallic,ceramic and/or polymeric origin, prior to the beginning offibrillogenesis. The fibrillogenesis is subsequently induced byincreasing the temperature. The fibrils formed in situ can either remainas a collagen gel, or be dried analogously to the surface coating.

The implant or scaffold prepared in this way can advantageously besterilized using the known nonthermal methods such as ethylene oxide orgamma irradiation and stored at room temperature.

The implant or scaffold coated according to the invention with anosteogenic matrix composite is delineated by the following advantagesfrom the solutions known from the prior art:

-   -   Good biological compatibility and functionality of the matrix        produced by means of largely physiological composition and        structure on account of the conditions in the production and use        of components which correspond to those of the natural cell        environment    -   High variability with respect to employable components and their        proportions in the component mixture    -   Easy storage and sterilization conditions    -   High specificity and efficiency due to the utilization of        endogenous osteogenic factors.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated in more detail by means of the followingworking examples, comparative tests and figures.

The figures show

FIG. 1 Influence of decorin and chondroitin sulfate, (CS) on theformation of collagen fibrils, measured as the increase in the turbidityof a fibrillogenesis solution in OD over time

FIG. 2 AFM photographs of the fibril structure

FIG. 3 Chondroitin sulfate and, decorin present in osteogenic matrixcomposites according to the invention

FIG. 4 Binding behavior of osteogenic matrix composites according to theinvention for the recombinant growth factors BMP-4 and TGF-1β

FIG. 5 Behavior of primary rat calvaria osteoblasts on variousosteogenic matrix composites according to the invention influence onadhesion and osteopontin expression

FIG. 6 Activity of alkaline phosphatase in rat calvaria cells on variousosteogenic matrix composites according to the invention after additionof 4 pmol/cm² of BMP-4

FIG. 7 New bone formation on the implant surface in percent after 6months in minipig jaw

DETAILED DESCRIPTION OF THE INVENTION WORKING EXAMPLE 1 Fibril Structureafter Fibrillogenesis under Various Conditions

For the generation of the osteogenic matrix composite, a solution ofcollagen monomers in 0.01 M acetic acid is prepared by stirring for 24hours at 4° C. The collagen fibrils are subsequently formed in thepresence of the noncollagenic components by a process ofself-aggregation (fibrillogenesis) in aqueous phosphate buffer solutionsat neutral pH and a temperature of 37° C.

The range for the formation of the fibrils is between 0.5 and 5 mg ofcollagen/ml and 0.1 to 5 mg of glycosaminoglycan/ml, 1 mg/ml of collagenand 0.2 mg/ml of GAG and 30 μg/ml of proteoglycan being the preferredconditions. The preferred fibrillogenesis parameters were a 30 mmol/lphosphate buffer pH 7.0, either with 135 mmol/l of NaCl or without NaCladdition.

Glycosaminoglycans or other matrix components are added to the collagenmonomers before fibrillogenesis and thereby integrated at leastpartially into the resulting fibrils in the following process offibrillogenesis.

FIG. 1 shows, in a measurement of the turbidity of a solution caused byfibril formation, over time, that increasing amounts of decorin(indicated in molar ratios) cause a slowing of the formation kineticsand a reduction of the maximum OD values, indicative of a reduction ofthe fibril diameter. For chondroitin sulfate, an opposite effect is tobe observed. Formation conditions: 250 μg/ml of collagen, 37° C., 30mmol/l of phosphate buffer pH 7.4 containing 135 mmol/l of NaCl.

In FIG. 2, the influence of the formation conditions on the structure ofthe resulting fibrils is documented in AFM photographs. Addition ofdecorin reduces the fibril diameter (a and d) under all conditions. Forchondroitin sulfate, in particular under conditions of low ionicstrength, a markedly more heterogeneous distribution of the fibrildiameter is visible with increase in the average fibril diameter (f),while the effect is not apparent at higher ionic strengths (c). b and eshow the fibril structure without noncollagenic additives. Formationconditions: 250 μg/ml of collagen, 37° C., 30 mmol./l of phosphatebuffer pH 7.4 (buffer A) or 30 mmol/l of phosphate buffer pH 7.4containing 135 mmol/l of NaCl (buffer B).

In all cases, however, during fibrillogenesis in vitro the collagenmonomers form the characteristic transversely striated fibrilsanalogously to the in vivo structures, the structure of the resultingfibrils being influenced both by the process parameters (pH, ionicstrength, phosphate concentration) and by the nature and amount of theadded noncollagenic components. Collagen fibrils containingnoncollagenic constituents such as glycosaminoglycans or decorin canaccordingly be produced in a comparatively wide range of mass ratios,within which the integration of the collagen into the fibrils is not oris only slightly influenced.

WORKING EXAMPLE 2 Incorporation of Noncollagenic Components intoCollagen Fibrils

For generation of the osteogenic matrix composite, a solution ofcollagen monomers in 0.01 M acetic acid is prepared by stirring at 4° C.for 24 hours. The collagen fibrils are subsequently formed by a processof self-aggregation (fibrillogenesis) in aqueous phosphate buffersolutions at neutral pH in the presence of the noncollagenic components.Formation conditions: 250 μg/ml of collagen, 37° C., 30 mmol/l ofphosphate buffer pH 7.4 (buffer A) or 30 mmol/l of phosphate buffer pH7.4 containing 135 mmol/l of NaCl (buffer B) with different chondroitinsulfate and decorin concentrations.

After washing and hydrolysis of the fibrils in 500 μl of 6 M HCl at 105°C. for 6 hours, decorin and chondroitin sulfate integrated into thefibrils was determined according to the method of Pieper et al. [PieperJ S, Hafmans T, Veerkamp J H, van Kuppevelt T H. Development oftailor-made collagen-glycosaminoglycan matrices: EDC/NHS crosslinking,and ultrastructural aspects. Biomaterials 2000; 21(6): 581-593].

For chondroitin sulfate, the extent of the integration is dependent onthe ionic strength of the buffer system. used. For low ionic strengths(buffer A), of the 20 μg employed, about 2.5 μg of CS are incorporatedon 250 μg of collagen, for high ionic strengths (buffer B), however,only a third of this amount (FIG. 3).

The incorporation of decorin also depends on the buffer system used. Forbuffer A, a third of the amount employed is incorporated, while thevalues for buffer B were again markedly lower.

WORKING EXAMPLE 3 Recruitment of Growth Factors by an Implant Coatedwith an Osteogenic Matrix Composite

Matrices composed and produced according to the invention can accelerateand improve bone formation and accumulation without the use ofrecombinant growth factors by the recruitment of endogenous growthfactors. In the experiment, such a binding behavior can only bedemonstrated using recombinant growth factors.

A sandblasted, cylindrical sample of TiAl6V4 having a diameter of 10 mmis cleaned with ethanol, acetone and water.

A solution of 1 mg/ml of bovine collagen type I in 0.01 M acetic acid isproduced by stirring overnight at 4° C. Noncollagenic ECM components(glycosaminoglycan 30 μg/ml, proteoglycans 15 μg/ml) are added to thissolution. The mixtures are treated with fibrillogenesis buffer (60mmol/l of phosphate, 270 mmol/l of NaCl, pH 7.4) on ice and incubated at37° C. for 18 h. The resulting fibrils are centrifuged off, washed,homogenized and resuspended to give a final concentration of 1 mg/ml.

The cylindrical sample is coated (dip-coating) with this solution at RTfor 15 min, washed with water and dried.

Subsequently, growth factors (recombinant BMP-4 or TGF-1β) areimmobilized on these -surfaces by an adsorption process (4° C., 18 h,from PBS) and subsequently. determined by means of ELISA.

These in vitro tests with recombinant growth factors show that by theaddition according to the invention of noncollagenic components, thebinding of the growth factors rhBMP-4 (in particular by addition ofchondroitin sulfate) or rhTGF-1β (in particular by addition of decorin)to the matrix is increased. For BMP, with small amounts (2-20 ng/cm²) noeffect is observed, with higher amounts (from 50 ng/cm²), however, anapproximately 10% higher binding to the chondroitin sulfate-containinglayer occurs, compared with the pure collagen layer, shown in % of theamount employed (FIG. 4).

For rhTGF-1β, increased binding is detectable on decorin-containingsurfaces both for 1 ng/cm² and for 10 ng/cm².

Formation conditions of the matrix: 500 μg/ml of collagen, 30 μg/ml ofdecorin and/or chondroitin sulfate, 37° C., 30 mmol/l of phosphatebuffer pH 7.4 containing 135 mmol/l of NaCl.

WORKING EXAMPLE 4 Investigations with Rat Calvaria Osteoblasts onVarious Matrix Composites

FIG. 5 shows the behavior of primary rat calvaria osteoblasts on variousmatrices. Initial adhesion of the cells to different matrix compositionswas analyzed by means of cell morphology, cytoskeletal organization(actin staining with phalloidin) and formation of the focal adhesioncomplexes by means of integrin receptors (immunostaining againstvinculin). Adhesion was most pronounced after 2 hours on collagen-CSmatrices followed by collagen-decorin. The formation of the FACS(green-yellow dots and red on the ends of the actin fibrils) was alsopromoted and accelerated by decorin and particularly CS. Controls usingpure collagen matrices showed significantly less FACS after 2 hours.

The influence of the matrix composition on the differentiation of theosteoblasts was investigated by means of the expression of the markerprotein osteopontin by means of fluorescence-activated cell scanning.Osteoblasts on collagen-CS surfaces produced 5 times more osteopontin(˜2500 fluorescence units) after 8 days than cells on pure collagensurfaces (−500 fluorescence units). Formation conditions of the matrix:500 μg/ml of collagen, 30 μg/ml of decorin and/or chondroitin sulfate,37° C., 30 mmol/l of phosphate buffer pH 7.4 containing 135 mmol/l ofNaCl.

Further investigations with rat calvaria osteoblasts showed differentcell reactions on rhBMP-4 depending on the composition of the carriermatrix. FIG. 6 shows the activity of the alkaline phosphatase inactivity units U per mg of protein after addition of 4 pmol/cm² ofrhBMP-4 to rat calvaria cells. On decorin-containing matrices, the BMPactivity is underregulated, while on chondroitin sulfate-containingmatrices it is increased. Formation conditions of the matrix: 500 μg/mlof collagen, 30 μg/ml of decorin and/or chondroitin sulfate, 37° C., 30mmol/l of phosphate buffer pH 7.4 containing 135 mmol/l of NaCl.

WORKING EXAMPLE 5 Animal Experiments

In animal experiments, it was surprisingly found that matrices providedwith recombinant growth factors perform markedly more poorly withrespect to induced bone formation than the noncrosslinked osteogenicmatrix composites according to the invention based on collagen type Iand chondroitin sulfate.

Ti implants, which have annular incisions at right angles to the axisand thus represent a defect model, are cleaned with 1% Triton X-100,acetone and 96% ethanol, rinsed with distilled water and dried.

The implants employed are coated in two successive dip-coating stepswith:

-   -   A. fibrils of collagen type I,    -   B. osteogenic matrix composite according to the invention based        on collagen type I and chondroitin sulfate according to working        example 1    -   C. osteogenic matrix composite according to the invention based        on. collagen type I and chondroitin sulfate according to working        example 1

The implants are washed with distilled water, air-dried and sterilizedwith ethylene oxide at 42° C. for 12 h. Immediately before implantation,the surface condition C is coated overnight with recombinant BMP-4 (400ng/ml) at 4° C. and subsequently dried.

The implants are employed in the lower jaw of minipigs. The bone implantcontact was determined histomorphometrically after 6 months.

The highest percentage for this contact is obtained for implants coatedwith the osteogenic matrix according to the invention based on collagenand chondroitin sulfate (27.8%), while implants with the same coatingand recombinant BMP-4 and the combination were around 15% and thusmarkedly lower. The lowest values are obtained for the pure collagencoating (12.8%) (FIG. 7).

The following abbreviations are used in the description of theinvention:

-   bFGF Basic fibroblast growth factor-   BMP Bone morphogenetic protein-   ECM Extracellular matrix-   EGF Endothelial growth factor-   FACITs Fibril associated collagen with interrupted triple helix-   FACS Focal adhesion contacts-   FGF Fibroblast growth factor-   GAG Glycosaminoglycan-   HAP Hydroxylapatite-   IGF-I Insuline-like growth factor-   PGA Polyglycolic acid-   PLA Polylactic acid-   SLRP Small leucine-rich protein-   TCP Tricalcium phosphate phases-   TES (N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid)-   TGF-β Transforming growth factor β-   VEGF Vascular endothelial growth factor-   WF Growth factor

1-13. (canceled)
 14. Osteogenic matrix composite of collagen and atleast one noncollagenic ECM component or its derivatives, wherein thecollagen component consists of noncrosslinked collagen fibrils producedby means of fibrillogenesis and in that i-o this are integrated the atleast one noncollagenic ECM component or its derivatives.
 15. Theosteogenic matrix composite as claimed in claim 14, wherein thenoncollagenic ECM components contain glycosaminoglycans.
 16. Theosteogenic matrix composite as claimed in claim 15, wherein thenoncollagenic ECM component contains chondroitin sulfate of type A, C,D, or E, dermatan sulfate, keratan sulfate, heparan sulfate, heparin,hyaluronic acid and their derivatives, individually or mixed.
 17. Theosteogenic matrix composite as claimed in claim 14, wherein thenoncollagenic ECM component contains noncollagenic matrix proteins. 18.The osteogenic matrix composite as claimed in claim 17, wherein thenoncollagenic ECM component contains, as noncollagenic matrix proteins,fibronectin, decorin, biglycan, laminin, versican individually or mixed.19. The osteogenic matrix composite as claimed in claim 14, wherein thecollagen component consists of one of the collagens I, II, III, V, IX,XI, or combinations thereof.
 20. A method for the production of anosteogenic matrix composite of collagen and at least one noncollagenicECM component or its derivatives, wherein collagen fibrils are producedby means of fibrillogenesis, in that before fibrillogenesis the at leastone noncollagenic ECM component or its derivatives is added, and in thatthe collagen fibrils produced in this way are resuspended in water or ina buffer system and optionally lyophilized.
 21. The method as claimed inclaim 20, wherein the flbrillogenesis is carried out under theconditions, temperature range from 4° C. to 40° C., preferably 25° C. to37° C., collagen concentration of 5C to 5000 pg/ml, preferably 250 to1000 pg/ml, pH 4 to pH 9, preferably pH 6 to pH 8, phosphate content upto 500 mmol/l, preferably 30 to 60 mmol/l, NaCl content up to 1000mmol/l, preferably up to 300 mmol/l.
 22. The use of an osteogenic matrixcomposite as claimed in claim 14 for the coating of implants orscaffolds for tissue engineering.
 23. A method for the production of animplant or of a scaffold for tissue engineering having a coating of anosteogenic matrix composite as claimed in claim 14, wherein collagenfibrils are produced by means of fibrillogenesis, in that beforefibrillogenesis the at least one noncollagenic ECM component or itsderivatives are added, and in that the collagen fibrils produced in thisway are resuspended in water or in a buffer and subsequently immobilizedon the surface of the implant or of the scaffold in a dip-coatingprocess.
 24. A method for the production of a scaffold for tissueengineering having a coating of an osteogenic matrix composite asclaimed in claim 14, wherein collagen fibrils are produced by means offibrillogenesis, in that before fibrillogenesis the at least onenoncollagenic ECM component or its derivatives are added in such a waythat fibril formation is induced in the scaffold, where the fibrilsformed in situ either remain as a gel or are dried.
 25. An implant orscaffold for tissue engineering having a coating of an osteogenic matrixcomposite as claimed in claim
 14. 26. A coating solution comprising anosteogenic matrix composite as claimed in claim 14.